Directed evolution of a regioselective halogenase for increased thermostability

Abstract

Compounds and methods are providing involving RebH variants with improved properties. directed evolution based on random mutagenesis was employed to generate a series of RebH variants. RebH variants with improved thermostability and increased activity at elevated temperatures were generated.

The invention claimed is: 
     
       1. An isolated RebH variant polypeptide of SEQ ID NO: 1 comprising at least one amino acid substitution, wherein the at least one amino acid substitution results in improved halogenating activity; and wherein the at least one amino acid substitution is selected from the group consisting of S2P, I52T, A58V, M71V, M71T, M71A, M71C, N75K, E96V, D101G, S110P, S110L, F111S, G112S, G112D, L113D, L113N, L114P, S130L, K145M, K145R, N166S, F171I, K187R, D203A, D203G, T213A, V225I, K237E, V256I, T258A, D264G, T283A, L289P, F312L, T322I, T348A, L380F, T394M, F396Y, F396L, R400C, T413A, E423D, A442V, S448P, L453P, F458S, F458L, E461G, F465C, F465L, N467T, N470S, A476T, A476V, V481A, Q494R, T496R, T496A, G504S, and R509Q, wherein the RebH variant polypeptide is at least 85% identical to SEQ ID NO:1. 
     
     
       2. The RebH variant of  claim 1  comprising the amino acid substitutions S2P, M71V, K145M, N467T, N470S, and G112S. 
     
     
       3. The RebH variant polypeptide of  claim 1 , wherein the polypeptide halogenates an aromatic substrate. 
     
     
       4. The RebH variant polypeptide of  claim 1 , wherein the polypeptide halogenates a substrate regioselectively. 
     
     
       5. The RebH variant polypeptide of  claim 1 , wherein the polypeptide displays improved thermostability over wild-type RebH. 
     
     
       6. The RebH variant polypeptide of  claim 1 , wherein the polypeptide displays increased halogenating activity at an elevated temperature. 
     
     
       7. The RebH variant polypeptide of  claim 1 , wherein the polypeptide halogenates the wild-type RebH native substrate tryptophan. 
     
     
       8. The RebH variant polypeptide of  claim 1 , wherein the polypeptide halogenates non-native substrates. 
     
     
       9. The RebH variant polypeptide of  claim 1 , wherein the polypeptide halogenates a substrate and wherein the substrate comprises a molecule selected from the group consisting of indole, tryptoline, 2-methyltryptamine, eleagnine, pinoline, tetrahydroharmine, debromodesformylflustrabromine, yohimbine, evodiamine, pindolol, carazolol, and carvedilol. 
     
     
       10. The RebH variant polypeptide of  claim 1 , wherein the polypeptide halogenates using a halogen selected from the group consisting of fluoride, chloride, bromide and iodide. 
     
     
       11. The RebH variant polypeptide of  claim 1 , wherein the polypeptide displays a prolonged catalyst lifetime. 
     
     
       12. The RebH variant polypeptide of  claim 1 , wherein the polypeptide displays an increased tolerance to proteolysis. 
     
     
       13. The RebH variant polypeptide of  claim 1 , wherein the polypeptide displays an increased tolerance to organic solvents. 
     
     
       14. The RebH variant polypeptide of  claim 1 , wherein the RebH variant polypeptide halogenates in the absence of a harsh chemical oxidant. 
     
     
       15. A RebH variant polypeptide comprising at least one amino acid substitution at position 2, 52, 58, 71, 75, 96, 101, 110, 111, 112, 113, 114, 130, 145, 166, 171, 187, 203, 213, 225, 237, 256, 258, 264, 283, 289, 312, 322, 348, 380, 394, 396, 400, 413, 423, 448, 453, 455, 458, 461, 465, 467, 470, 476, 481, 494, 496, 504 and/or 509 in SEQ ID NO:1, wherein the RebH variant polypeptide is at least 85% identical to SEQ ID NO:1. 
     
     
       16. The isolated RebH variant polypeptide of  claim 1 , wherein the variant comprises at least a S2P substitution.